This invention relates to a rapid, sensitive method for detecting the presence of antibiotics in liquids such as milk, body fluids, meat extracts, and fermentation broths.
The ability to detect small concentrations of antibiotics in liquids is important in various situations. One example is the food industry where the use of antibiotics in the treatment of animals which produce food stuffs has created a need for a rapid, accurate test method which can be used in the field by bulk food handlers and the like. Because penicillins are used to treat mastitis in dairy cattle, and because the Food and Drug Administration restricts the penicillin content of milk, antibiotic detection methods suitable for rapidly and accurately screening milk are particularly important. Thus, the growing medical concern about ingestion of small amounts of antibiotics by humans is directing attention to the incidence of penicillin in milk at levels in the range of 0.010-0.050 I.U./ml or greater and to simple screening methods for detecting such minute quantities of penicillin and other antibiotics.
At the present time, there are a variety of known antibiotic detection procedures. Most of these involve microbiological techniques wherein the presence or absence of penicillin or other antibiotic is determined by observing the inhibition of growth of antibiotic sensitive microorganisms in the pesence of the test sample. Formerly, such procedures required incubation times of four hours or more, but by employing microorganism strains "supersensitive" to antibiotics, the time required to perform the assays has been reduced to between 2 and 21/2 hours.
Other antibiotic detection techniques exploit various other unique properties of the antibiotic or class of antibiotics to be detected as a test basis. Thus, in Immobilized Enzyme-Based Flowing-Stream Analyzer for Measurement of Penicillin in Fermentation Broths, J. F. Rusling et al., Analytical Chemistry, Vol. 48, p. 1211, (July, 1976), a test based on the enzymatic hydrolysis of pencillin with an immobilized .beta. lactamase derivative is disclosed. Simple Ultrasensitive Test for Detecting Penicillin in Milk, J. M. A. Palmer et al., J. Dairy Science, Vol. 50, p. 1390, discloses a penicillin detection method based on the growth of Bacillus subtilis spores on nutrient-spore-dye paper discs residing in a small sample of milk exposed to the air. As the water content of the milk evaporates, penicillin concentration, if any, increases and induces a color change in the dye which is indicative of concentration. U.S. Pat. No. 3,586,483 to J. G. Heider et al. discloses a method of detecting tetracycline antibiotics in fluids by absorbing the fluid on an absorbent strip containing a complexing metal which forms a fluorescent metal complex with the antibiotic, and by observing the fluorescence of the metal complex under ultraviolet light.
The foregoing and other available detection techniques vary widely with respect to their sensitivity and speed. It is believed that no presently available test is capable of detecting as little of 0.01 I.U. of penicillin per milliliter (6 ng/ml) in less than an hour. If such a test were available, it would become economical to rapidly and reliably determine, for example, whether milk sampled in the field from relatively small batches contained antibiotic concentrations in excess of Food and Drug Administration standards.